Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos

Clare Hudson; Cathy Sirour; Hitoyoshi Yasuo

doi:10.7554/eLife.14692

. 2016 Jun 28;5:e14692. doi: 10.7554/eLife.14692

Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos

Clare Hudson ^1,^*,^†, Cathy Sirour ¹, Hitoyoshi Yasuo ^1,^*,^†

Editor: Alejandro Sánchez Alvarado²

PMCID: PMC4945153 PMID: 27351101

Abstract

In many bilaterian embryos, nuclear β-catenin (nβ-catenin) promotes mesendoderm over ectoderm lineages. Although this is likely to represent an evolutionary ancient developmental process, the regulatory architecture of nβ-catenin-induced mesendoderm remains elusive in the majority of animals. Here, we show that, in ascidian embryos, three nβ-catenin transcriptional targets, Foxa.a, Foxd and Fgf9/16/20, are each required for the correct initiation of both the mesoderm and endoderm gene regulatory networks. Conversely, these three factors are sufficient, in combination, to produce a mesendoderm ground state that can be further programmed into mesoderm or endoderm lineages. Importantly, we show that the combinatorial activity of these three factors is sufficient to reprogramme developing ectoderm cells to mesendoderm. We conclude that in ascidian embryos, the transient mesendoderm regulatory state is defined by co-expression of Foxa.a, Foxd and Fgf9/16/20.

DOI: http://dx.doi.org/10.7554/eLife.14692.001

Research Organism: C. intestinalis

Introduction

The mesoderm, endoderm and ectoderm arise during embryonic development by a process termed germ layer segregation. In many species, at least part of the endoderm and mesoderm derive from transient ‘mesendoderm’ precursors, as is the case in ascidian embryos (Kimelman and Griffin, 2000; Rodaway and Patient, 2001). However, the precise nature of this induced regulatory state is not well understood. In ascidians, the first animal-vegetal (A-V) oriented cell division generates the eight-cell stage embryo and segregates the mesendoderm and some neural lineages into two pairs of vegetal founder lineages (the A- and B-line) and the ectoderm (epidermis and neural) into two pairs of animal lineages (a- and b-line) (Conklin, 1905; Nishida, 1987). This study focuses on the A-line mesendoderm lineages. From the 8- to 16-cell stage, the two A4.1 blastomeres divide medio-laterally to generate the two pairs of neuro-mesendodermal NNE cells, for notochord/neural/endoderm (Figure 1a). NNE cells then divide along the A-V axis to generate NN cells (notochord/neural) and E cells (mostly endoderm) at the 32-cell stage (Figure 1a). Subsequently, NN cells segregate into notochord and neural lineages at the 64-cell stage. At this stage, while the medial E cell generates two endoderm precursors, the lateral-most E cell is subject to an inductive interaction resulting in the generation of one endoderm and one mesoderm (the trunk lateral cell lineage) precursor (Shi and Levine, 2008). Later, during neural plate patterning, a muscle precursor is also generated from the lateral borders of the NN-lineage-derived neural plate (Nicol and Meinertzhagen, 1988; Nishida, 1987). Thus, as in other species, ascidian germ layer segregation can be viewed as a progressive process with part of the neural tissue arising from bipotential neuro-mesodermal progenitors (Henrique et al., 2015; Tzouanacou et al., 2009). The earliest cell divisions of the ascidian embryo along the A-V axis at the 8- and 32-cell stages can be considered as the earliest steps of germ layer segregation.

Figure 1. — (a) Schematic drawings of embryos at the 16- and 32-cell stages. In this and all subsequent figures, where shown, a green dashed line separates the animal (ectoderm) from the vegetal (mesendoderm) hemispheres and a brown dashed line separates A- (A4.1) and a- (a4.2) lineages from B- (B4.1) and b- (b4.2) lineages. Different embryonic founder lineages are indicated on the drawings. NN and E cells are indicated in red and blue, respectively. Below the embryo drawings is a schematic representation of the two rounds of nβ-catenin-driven binary fate decisions that segregate firstly the mesendoderm lineages from the ectoderm lineages at the 16-cell stage and secondly segregate the mesoderm (NN) lineages from the endoderm (E) lineages at the 32-cell stage (Hudson et al., 2013). (b, c) Embryos analysed at the 16-cell stage for the marker indicated to the left of the panels following the treatment indicated above the panels [(b) vegetal pole view; (c) lateral view, vegetal pole to the right]. The numbers on the bottom-left corner of each panel indicate the proportion of embryos that the panel represents. The posterior most cells (at the bottom of the panels) are transcriptionally quiescent cells that will generate the germ line (Shirae-Kurabayashi et al., 2011). For *Foxa.a* expression in (b) control embryos showed expression in all four A-line (NNE) cells in 34/34 embryos, and in B-line cells, in 15/34 embryos, as indicated, whereas β-catenin-MO (β-cat-MO) injected embryos showed expression in NNE cells (31/31), but not B-line (0/31). Expression of *Foxa.a* in the four a-line precursors (not visible in the image) was not affected by β-catenin-MO injection.

**DOI:** http://dx.doi.org/10.7554/eLife.14692.002

β-catenin is a transcriptional co-activator which acts in a complex with TCF DNA-binding proteins to mediate the canonical Wnt signalling pathway (Valenta et al., 2012). The β-catenin/TCF complex promotes endoderm or mesendoderm in a wide range of organisms, and this process is therefore likely to represent an ancestral mechanism (Darras et al., 2011; Henry et al., 2008; Hudson et al., 2013; Imai et al., 2000; Logan et al., 1999; McCauley et al., 2015; Miyawaki et al., 2003; Momose and Houliston, 2007; Wikramanayake et al., 1998, 2003). We have previously shown that the earliest steps of germ layer segregation in ascidian embryos are mediated by two rounds of nuclear(n)-β−catenin-dependent binary fate decisions. The first nβ-catenin-driven binary fate decision takes place at the 8- to 16-cell stage. During this process, the β-catenin/TCF complex is differentially activated between mesendoderm and ectoderm progenitors, resulting in segregation of these lineages (Figure 1a) (Hudson et al., 2013; Oda-Ishii et al., 2016; Rothbächer et al., 2007). The second step takes place at the 32-cell stage and controls the segregation of NNE mesendoderm cells into endoderm (E cell) and notochord/neural (NN cell) lineages (Hudson et al., 2013). During this step, the β-catenin/TCF complex is again differentially activated between E and NN cells (Figure 1a). Therefore, cells in which nβ-catenin remains active during the two steps (ON + ON) are specified as endoderm lineage, cells in which nβ-catenin remains inactive during the two steps (OFF + OFF) are specified as ectoderm lineage and cells in which nβ-catenin is active during the first step but inactive during the second step (ON + OFF) are specified as notochord-neural lineage (Hudson et al., 2013). These two rounds of nβ-catenin-driven switches result in transcriptional activation of the lineage specifiers, Zic-related.b (Zic-r.b, formally ZicL) and Lhx3/4 (formally Lhx3), in NN and E cells, respectively (Imai et al., 2002c; Satou et al., 2001). One of the key features of these reiterative nβ-catenin-driven binary fate decisions is that the same asymmetric cue (nβ-catenin) is interpreted differently during each step (Bertrand and Hobert, 2010). Thus, in the NNE lineage, it is likely that the transient regulatory state induced by the first nβ-catenin input in NNE cells confers a distinct transcriptional response to the second nβ-catenin input on E cells.

In this study, we characterise the NNE lineage specification factors, which are induced by the first nβ-catenin input and address how these mesendoderm factors feed into the gene regulatory network of the NN and E lineages.

Results

Foxa.a, Foxd and Fgf9/16/20 are nβ-catenin transcriptional targets in NNE cells

Following the first nβ-catenin activation at the 16-cell stage, Foxa.a, Foxd, Fgf9/16/20, cadherinII and βCD1 (β-catenin downstream gene 1) are induced in the NNE cells, with at least Foxd and Fgf9/16/20 being direct targets of the β-catenin/Tcf7 complex (Imai, 2003; Imai et al., 2002a, 2002b, 2002c; Kumano et al., 2006; Oda-Ishii et al., 2016; Rothbächer et al., 2007; Satou et al., 2001). Consistent with a recent study (Oda-Ishii et al., 2016), we confirmed that in β-catenin–inhibited (β-catenin-MO injected) embryos analysed at the 16-cell stage, Foxd and Fgf/9/16/20 expression was lost (Figure 1b). In addition to the mesendoderm lineages, Foxa.a is also expressed in the a-line anterior ectoderm lineages in a nβ-catenin-independent fashion (Figure 1b,c) (Lamy et al., 2006). In β-catenin–inhibited embryos, Foxa.a expression persisted in NNE and a-lineage cells, probably due to transformation of vegetal cells into animal cells that has been reported previously (Figure 1b) (Imai et al., 2000; Oda-Ishii et al., 2016). Conversely, ectopic stabilisation of nβ-catenin resulted in activation of all three genes in ectoderm lineages at the 16-cell stage (Figure 1c). This was achieved by treating embryos with BIO, a chemical inhibitor of the upstream inhibitory regulator of β-catenin, GSK-3, from the eight-cell stage (Meijer et al., 2003). Thus, our results confirm that Foxd, Foxa.a and Fgf9/16/20 are transcriptional targets of nβ-catenin in vegetal cells, although Foxa.a also has a nβ-catenin-independent expression in a-line animal cells.

Foxa.a, Foxd and Fgf9/16/20-signals are required for the correct initiation of both NN and E gene expression

It is likely that these gene products, activated by the first nβ-catenin signal in NNE cells, act together with the second differential nβ-catenin signal to activate the distinct gene regulatory networks between NN and E cells. Consistent with this idea, Foxa.a has been shown to be required for both NN lineage and endoderm gene expression (Imai et al., 2006), with Foxd specifically required for NN lineage, but not endoderm fates, and Fgf9/16/20 contributing to notochord induction from the NN lineage (Imai et al., 2002a, 2002b; Yasuo and Hudson, 2007). However, we found that inhibiting any one of these factors prevented the correct initiation of gene expression in both NN (Zic-r.b) and E (Lhx3/4) lineages (Figure 2a, Table 1). We inhibited these factors using Morpholino anti-sense oligonucleotides (Foxd-MO, Foxa.a-MO, Fgf9-MO) and analysed Zic-r.b and Lhx3/4 expression at the 32-cell stage, when NN and E cell lineages become segregated. FGF signals are frequently mediated by the MEK/ERK signalling pathway, leading to transcriptional activation via ETS family transcription factors, as is the case in ascidian embryos (Bertrand et al., 2003; Kim and Nishida, 2001; Miya and Nishida, 2003; Yasuo and Hudson, 2007). We confirmed that Fgf9/16/20 is responsible for the broad activation of ERK at the 32-cell stage in most vegetal lineages, including NN and E lineages, as well as two neural lineages in the ectoderm (Figure 2—figure supplement 1f). Treatment of embryos from the 16-cell stage with the MEK inhibitor U0126, also inhibits this ERK1/2 activation (Kim and Nishida, 2001; Picco et al., 2007). Inhibition of Fgf9/16/20, MEK or ETS1/2 (ETS1/2-MO) gave similar results, although inhibition of ETS1/2 gave only a weak down-regulation of Zic-r.b expression at the 32-cell stage, perhaps indicating the involvement of additional transcription factors that are also known to mediate FGF signals in Ciona embryos (Figure 2a; Table 1) (Bertrand et al., 2003; Gainous et al., 2015). Maintenance of Foxa.a, Foxd and Fgf9/16/20 expression at the 32-cell stage is independent of each other (Figure 2—figure supplement 1a), as was shown previously for Foxd and Fgf9/16/20 in Ciona savigni embryos (Imai et al., 2002a).

Figure 2. — (a) Embryos analysed at the 32-cell stage. The marker analysed is indicated on the left of the panels and the treatment indicated above the panels. The average number of NN (*Zic-r.b*) or E (*Lhx3/4*) cells expressing detectable levels of each gene is indicated. This remaining expression was generally weaker than control level expression. ‘n=’ represents the number of embryos analysed. (b) Expression of *Efna.d* under the conditions indicated. Embryos are shown in notochord-side view, animal pole up. The graph shows the average number of cells expressing *Efna.d* in different vegetal lineages at the 32-cell stage, as indicated by the key. All embryos showed ectoderm expression. The number of embryos analysed is indicated above the bars on the graph.

**DOI:** http://dx.doi.org/10.7554/eLife.14692.003

Figure 2—figure supplement 1. — (a) Embryos analysed at the 32-cell stage. The marker analysed is indicated on the left of the panels and the treatment indicated above the panels. The average number of NN (*Zic-r.b*) or E (*Lhx3/4*) cells expressing detectable levels of each gene is indicated. This remaining expression was generally weaker than control level expression. ‘n=’ represents the number of embryos analysed. (b) Expression of *Efna.d* under the conditions indicated. Embryos are shown in notochord-side view, animal pole up. The graph shows the average number of cells expressing *Efna.d* in different vegetal lineages at the 32-cell stage, as indicated by the key. All embryos showed ectoderm expression. The number of embryos analysed is indicated above the bars on the graph.

**DOI:** http://dx.doi.org/10.7554/eLife.14692.003

Table 1.

Expression of Zic-r.b in NN cells and Lhx3/4 in E cells of 32-cell stage embryos, following inhibition of Fgf-signalling components.

DOI: http://dx.doi.org/10.7554/eLife.14692.006

	Control	U0126	ETS1/2-MO
Zic-r.b NN cell	4.0 cells (n = 163)	0.75 cells (n = 58)	3.4 cells* (n = 74)
Lhx3/4 E cell	3.9 cells (n = 153)	2.1** cell (n = 45)	0.7 cells (n = 92)

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* 44/74 embryos exhibited weaker levels of Zic-r.b expression compared to controls.

**Remaining expression was weaker than control levels of expression.

In FGF-inhibited embryos, Zic-r.b expression recovered at the 64-cell stage (Figure 2—figure supplement 1a) (Imai et al., 2006; Kumano et al., 2006). Zic-r.b expression at the 32- and 64-cell stages can be mediated by separate enhancer elements (Anno et al., 2006). In addition, in the NN-cell lineage, FGF-signalling is required for notochord fate, but has to be attenuated for neural fate (Minokawa et al, 2001; Picco et al., 2007; Yasuo and Hudson, 2007). Thus, an FGF-independent expression of Zic-r.b at the 64-cell stage, at least in neural fated cells, is not unexpected. In Foxa.a- and Foxd- inhibited embryos, Zic-r.b continues to be repressed in the NN-cell lineages at the 64-cell stage (Figure 2—figure supplement 1a) and later (Imai et al., 2006), consistent with a requirement for Foxa.a and Foxd for both NN cell lineage-derived structures, the notochord and caudal central nervous system (CNS) (Imai et al., 2002b, 2006).

Endoderm gene expression was continuously reduced up to at least the early gastrula stage, following inhibition of any one of the NNE factors (Figure 2—figure supplement 1a–e). However, using alkaline phosphatase activity as an indicator of endoderm formation (Whittaker, 1977), a complete loss of endoderm at larval stages was observed only in Foxa.a-inhibited embryos, consistent with previous studies (Figure 2—figure supplement 2) (Imai et al., 2002b, 2006). In Foxd and FGF-signal inhibited embryos, a large domain of alkaline phosphatase activity could be detected, suggesting that endoderm fate recovers in these embryos (Figure 2—figure supplement 2). Simultaneous repression of Foxd and FGF-signalling, however, resulted in both a stronger repression of early endoderm gene expression as well as an almost complete absence of alkaline phosphatase activity at larval stages (Figure 2—figure supplement 1–2). Thus, for eventual endoderm formation, the embryo is able to compensate for loss of either Foxd or FGF-signals but is not able to compensate for loss of both.

As well as promoting vegetal ‘mesendoderm’ fates, nβ-catenin also represses the ectoderm gene programme in vegetal cells (Hudson et al., 2013; Imai et al., 2000; Oda-Ishii et al., 2016; Rothbächer et al., 2007). In β-catenin knock-down embryos, ectopic expression of the early ectoderm gene Efna.d (formally ephrin-Ad) is observed in both NN and E cells at the 32-cell stage (Figure 2b) (Hudson et al., 2013) as well as in NNE cells at the 16-cell stage (Oda-Ishii et al., 2016). Double inhibition of Foxd and FGF-signals also resulted in ectopic expression of Efna.d in NN cells, but never in E cells (Figure 2b). Thus, NNE factors repress the ectoderm genetic programme in NN cells. A lack of derepression in E cells is probably due to the presence of nβ-catenin in E cells at the 32-cell stage (Hudson et al., 2013), suggesting that nβ-catenin can repress the ectoderm genetic programme both via and independently of the NNE factors.

Combinatorial activity of Foxa.a, Foxd and Fgf9/16/20 induces a mesendoderm state

Our data so far show that Foxa.a, Foxd and Fgf9/16/20-ERK1/2 are individually required for the correct initiation of the genetic programmes of both NN and E cell lineages. Indeed, co-expression of these three factors takes place only in mesendoderm lineages, the NNE and B-line mesendoderm lineages of the 16- and 32-cell stage embryo (Figure 1; Figure 2—figure supplement 1a). At the 32-cell stage, the E cells continue to express these three genes, while NN cells express only Foxa.a and Fgf9/16/20. However, we have previously shown that Foxd transcripts preferentially segregate into the NN cells during the NN-E cell division, before they rapidly disappear (Hudson et al., 2013). Thus, NN cells also contain Foxd transcripts early in their cell cycle. We conclude that Foxa.a, Foxd and Fgf9/16/20 are co-expressed only in mesendoderm lineages. We next addressed whether these three factors were sufficient to induce a mesendoderm regulatory state.

As well as in vegetal cells, Foxa.a is also expressed in a-line anterior animal cells and ERK1/2 is activated in one pair of a-line cells (the a6.5 pair) at the 32-cell stage (Figure 3a) (Hudson et al., 2003; Lamy et al., 2006). Thus, a6.5 cells possess two of the three mesendoderm lineage specifiers and yet they do not adopt an NNE-like lineage. Consistent with the notion that coexpression of Foxa.a, Foxd and Fgf9/16/20 represents a NNE regulatory state, reintroduction of the remaining factor, Foxd, by mRNA injection, was able to convert a-line cells to a mesendoderm state (Figure 3). Injection of Foxd mRNA resulted in ectopic expression of Zic-r.b in a-line cells at the 32-cell stage (Figure 3b) (Imai et al., 2002c). Similarly, expression of Bra, a marker of notochord precursors, was induced at the 64-cell stage (Figure 3b). The broad ectopic expression of Zic-r.b and Bra in the a-lineage was clearly not restricted to the a6.5 cells. The most likely reason for this was that Foxd mRNA injection led to weak activation of Fgf9/16/20 and strong inhibition of Efna.d expression in the ectoderm cells (Figure 3—figure supplement 1). Efna.d is a known antagonist of FGF-signals in Ciona and its inhibition results in widespread activation of ERK1/2 in ectoderm lineages (Ohta and Satou, 2013; Picco et al., 2007; Shi and Levine, 2008). Consistent with this, when Foxd mRNA-injected embryos were treated with U0126, the ectopic expression of Zic-r.b was reduced and Bra expression was completely suppressed, mimicking the effect of MEK inhibition on endogenous Zic-r.b and Bra gene expression (Figure 3b). Thus, injection of Foxd mRNA is sufficient to convert Foxa.a/ERK1/2-positive a-line cells into mesoderm.

Figure 3—figure supplement 1. — (a) Schematics show endogenous ectodermal expression of *Foxa.a, Foxd* (no expression) and activation of ERK (dpERK), indicated by blue dots. (**b–c**) Treatment is indicated above the panels and marker analysed to the left. All embryos in animal pole view except control *Bra* (vegetal pole view). Numbers show the total average number of cells per embryo expressing each marker. n = total number of embryos analysed. The graphs show the average number of cells expressing each maker in the lineages indicated on the keys, following the treatments indicated of the x-axis. No *Zic-r.b* expression was detected in endoderm lineages. In (c), the green arrowheads highlight the eight a-lineage cells. For (b), representative panels of uninjected/UO-treated and uninjected/BIO-treated embryos are not shown. The numbers of these experiments are: for *Zic-r.b*- U0126 alone n = 40 (average number of cells 2.6), BIO-16 alone n = 40 (average number of cells 2.9) and for *Bra*- U0126 alone, n = 39 (average number of cells 0.0); BIO-16 alone n = 31 (average number of cells 0.0).

**DOI:** http://dx.doi.org/10.7554/eLife.14692.007

Injection of Foxd mRNA, however, was not sufficient to induce endoderm gene expression in ectoderm cells (Figure 3c). This was expected since the two-step nβ-catenin binary fate decision model predicts that segregation of the endoderm lineage from the NNE lineage requires a second round of nβ-catenin activation (Figure 1a) (Hudson et al., 2013). Accordingly, when Foxd-mRNA injected embryos were treated from the late 16-cell stage with BIO in order to mimic the second input of nβ-catenin activation, both Zic-r.b and Bra were repressed and Lhx3/4 ectopically activated in the a-line ectoderm cells (Figure 3b–c). Thus, the NNE-like state induced in animal cells by Foxd mRNA injection behaves in the same way as NNE state of unmanipulated embryos.

Taken together, these experiments provide strong evidence that the combinatorial activity of Foxa.a, Foxd and Fgf9/16/20-ERK1/2 represents a NNE mesendoderm regulatory state downstream of the first round of nβ-catenin input. To further test this model, we addressed whether co-expression of Foxa.a, Foxd and Fgf9/16/20 was sufficient to rescue mesoderm in β-catenin-knockdown embryos. β-catenin-MO injected embryos would express only Foxa.a among the three genes (Figure 1b). We have shown that injection of Foxd mRNA results in induction of low levels of Fgf9/16/20 expression, together with a strong suppression of Efna.d expression (Figure 3—figure supplement 1). Thus, injection of Foxd mRNA should be sufficient to recapitulate a Foxd/Foxa.a/Fgf9/16/20 overlap. Consistent with this, injection of Foxd mRNA was able to rescue expression of NN-lineage genes (Zic-r.b and Bra) in β-catenin-MO embryos and, as expected, this recovery depended on an intact FGF-signalling pathway (Figure 4). We conclude that co-expression of Foxd+Foxa.a+Fgf9/16/20 is sufficient to induce a mesendoderm regulatory state, which can then be further programmed into mesoderm or endoderm lineage by manipulation of nβ-catenin activity.

Figure 4. — (**a–b**) Treatment is indicated above the panels and marker analysed to the left of the panels. The total average number of cells per embryo is indicated, ‘n=’ indicates the total number of embryos analysed for each treatment. The graphs show the average number of cells expressing each marker in the lineages indicated by the keys, following the treatments indicated.

**DOI:** http://dx.doi.org/10.7554/eLife.14692.009

Foxa.a, Foxd and Fgf9/16/20 act synergistically to reprogramme developing ectoderm cells to a mesendoderm state

We next addressed whether ectopic expression of Foxd, Foxa.a and Fgf9/16/20 was able to reprogramme developing ectoderm to a mesendoderm state (Figure 5, Figure 5—figure supplement 1). The upstream regulatory sequences of the Fucosyltransferase-like (FT) gene become active in ectoderm cells from the 64-cell stage, when the ectoderm genetic programme is already underway (Figure 5—figure supplement 1) and when these cells no longer express Foxa.a, Foxd or Fgf9/16/20 (Imai et al., 2004; Pasini et al., 2012). Using FT promoter-driven constructs (pFT>Foxa.a, pFT>Foxd and pFT>Fgf9/16/20), we expressed Foxa.a, Foxd and Fgf9/16/20 in different combinations in ectoderm lineages. To simplify the analysis and to rule out the possibility that signals from the vegetal cells may influence the experimental outcome, animal hemispheres of electroporated embryos were isolated by micro-dissection at the eight-cell stage. Isolated explants were cultured until the neurula stage when they were assayed for Bra expression (Figure 5a–b). Bra was chosen for this assay for its mesoderm (notochord)-specific expression. We observed a clear combinatorial effect between Foxa.a, Foxd and Fgf9/16/20 on the reprogramming of ectoderm to mesoderm, with strong induction of Bra seen only when all three constructs were co-electroporated (Figure 5b). This reprogramming was accompanied by a strong downregulation of ectoderm gene expression and ectopic expression of Zic-r.b in the ectoderm cells of whole embryos (Figure 5—figure supplement 1). Furthermore, ectoderm explants could be reprogrammed to adopt an endoderm state (Figure 5c). To achieve this, ectoderm explants from embryos electroporated with the triple combination (pFT>Foxa.a, pFT>Foxd and pFT>Fgf9/16/20) were treated with a pulse of BIO from the 76-cell stage to mimic the second round of nβ-catenin activation that normally drives the segregation of NN and E lineages (Figure 5c). The 76-cell stage was chosen, as at this stage ectopic expression driven by the pFT constructs is readily detectable (Figure 5—figure supplement 1a), the ectoderm programme is downregulated (Figure 5—figure supplement 1b) and ectopic Zic-r.b is not yet detected (Figure 5—figure supplement 1c). This stage thus represented the best approximation of the NNE state of normal embryos. We confirmed that BIO-treatment was able to induce nuclear translocation of β-catenin in isolated explants at the 76-cell stage (Figure 5—figure supplement 2). Endoderm induction was assayed by detection of Lhx3/4 expression at the mid-gastrula stage, that is approximately 1 hr after the onset of BIO-treatment. Coupling these three factors with BIO-treatment resulted in strong induction of Lhx3/4 expression. We conclude that the combinatorial activity of Foxa.a, Foxd and Fgf9/16/20 is sufficient to reprogramme developing ectoderm cells to adopt a mesendoderm state.

Figure 5. — (a) Experimental scheme. Embryos were electroporated and the ectoderm lineage (animal cap) isolated at the eight-cell stage. Ectodermal explants were cultured until the mid-gastrula stage for *Lhx3/4* expression or until the neurula stage for *Bra* expression. Optionally, explants were treated with BIO, when control sibling embryos reached the 76-cell stage, for approximately 1 hr prior to fixation (*Lhx3/4* only). (**b–c**) Expression of *Bra* (b) and *Lhx3/4* (c) in isolated ectodermal explants, following the treatments indicated above the panels. ‘n=’ represents the number of explants analysed. Graphs shows the percentage of explants with any level of *Bra* expression or level of *Lhx3/4* expression indicated by the key, under various conditions (*Foxa.a*= pFT>*Foxa.a*; *Foxd* = *pFT>Foxd; Fgf9= pFT>Fgf9/16/20*; control = unelectroporated).

**DOI:** http://dx.doi.org/10.7554/eLife.14692.010

Figure 5—figure supplement 1. — (a) Experimental scheme. Embryos were electroporated and the ectoderm lineage (animal cap) isolated at the eight-cell stage. Ectodermal explants were cultured until the mid-gastrula stage for *Lhx3/4* expression or until the neurula stage for *Bra* expression. Optionally, explants were treated with BIO, when control sibling embryos reached the 76-cell stage, for approximately 1 hr prior to fixation (*Lhx3/4* only). (**b–c**) Expression of *Bra* (b) and *Lhx3/4* (c) in isolated ectodermal explants, following the treatments indicated above the panels. ‘n=’ represents the number of explants analysed. Graphs shows the percentage of explants with any level of *Bra* expression or level of *Lhx3/4* expression indicated by the key, under various conditions (*Foxa.a*= pFT>*Foxa.a*; *Foxd* = *pFT>Foxd; Fgf9= pFT>Fgf9/16/20*; control = unelectroporated).

**DOI:** http://dx.doi.org/10.7554/eLife.14692.010

Discussion

In this study, we have identified Foxa.a, Foxd and Fgf9/16/20 as the mesendoderm lineage specifiers of the NNE cell. Transcriptional activation of Foxa.a, Foxd and Fgf9/16/20 is induced by the first nβ-catenin switch (Figures 1a,6a). Co-expression of these three factors is sufficient to reprogramme ectoderm cells to adopt a mesendoderm state. This ectopic mesendoderm state can be further converted into either mesoderm or endoderm by modulating nβ-catenin activation.

A model for ascidian germ layer segregation

We propose the following model to summarise the initial stages of germ layer segregation in ascidian embryos (Figure 6a). At the 8- to 16-cell stage of development, nβ-catenin, activated specifically in vegetal cells by as yet unknown mechanisms, promotes Foxa.a, Foxd and Fgf9/16/20 expression and represses ectoderm gene expression (Hudson et al., 2013; Imai et al., 2000; Oda-Ishii et al., 2016; Rothbächer et al., 2007). Foxa.a, Foxd and Fgf9/16/20, are co-expressed exclusively in mesendoderm lineages at the 16- to 32-cell stage of development (Imai et al., 2002a, 2002b; Oda-Ishii et al., 2016), where they are required, individually, for the correct initiation of both NN and E cell lineage gene expressions at the 32-cell stage (Figure 2a). The NNE factors are also required to repress ectoderm gene expression: co-inhibition of Foxd and Fgf9/16/20 resulted in ectopic ectoderm gene expression in NN cells (Figure 2b) and Foxd overexpression alone was able to repress ectoderm gene expression (Figure 3—figure supplement 1). However, our data also suggest that nβ-catenin can repress ectoderm gene expression independently of these three factors (Figure 2b). Recently, it has been shown that this can take place via a physical interaction between β-catenin/Tcf7 and Gata.a, preventing this key regulator of ectoderm lineage from binding to its DNA target sites (Oda-Ishii et al., 2016; Rothbächer et al., 2007).

Following inhibition of Foxa.a, Foxd or Fgf9/16/20, both endoderm and mesoderm development is perturbed at later stages of development, although there is a redundancy between Foxd and FGF-signalling for the eventual recovery of endoderm (Figure 2 and Figure 2—figure supplement 1, Figure 2—figure supplement 2) (Imai et al., 2002a, 2002b, 2002c, 2006; Kumano et al., 2006). It is likely that these factors play on-going roles during mesoderm and endoderm lineage progression. For example, ERK1/2 activity is detected in both notochord and endoderm until the early gastrula stage (Nishida, 2003; Yasuo and Hudson, 2007), Fgf9/16/20 is required at the 64-cell stage for induction of notochord and repression of neural gene expression in the notochord lineage (Imai et al., 2002a; Kim and Nishida, 2001; Minokawa et al., 2001; Yasuo and Hudson, 2007) and Foxa.a is continuously expressed in notochord and endoderm, suggesting an on-going role for Foxa.a in both of these lineages (Imai et al., 2004).

Importantly, creating ectopic zones of co-expression of these three factors in distinct embryological settings, revealed their strong synergistic ability to induce a mesendoderm state, which can be further programmed to an NN or E-like lineage by modulation of nβ-catenin levels (Figures 3–5 and Figure 3—figure supplement 1, Figure 5—figure supplement 1, Figure 5—figure supplement 2). We conclude that Foxa.a, Foxd and Fgf9/16/20 are crucial for the mesendoderm ground state that canalises the daughter lineages to adopt either E or NN fates depending on the status of the second nβ-catenin input.

It is important to bear in mind that the germ layers are still not fully segregated at the 32-cell stage. While this manuscript has focused on the mesendoderm fates that arise from the NNE lineage, this lineage also produces neural tissue. NNE cells divide into E cells and NN cells. In addition to notochord, the NN cell generates the posterior part of the CNS, including the equivalent of the ‘spinal cord’ of vertebrates (reviewed in [Hudson, 2016]). The binary cell fate decision between neural and notochord takes place at the 64-cell stage (Minokawa et al., 2001; Picco et al., 2007). The lateral neural progenitors that arise from the NN-cell lineage also produce a muscle cell during neural plate patterning, following another neuromesodermal binary fate decision (reviewed in [Hudson and Yasuo, 2008]). Bipotential neuromesoderm progenitors are not an ascidian novelty (Henrique et al., 2015; Tzouanacou et al., 2009). For example, in the zebrafish tailbud, bipotential neuromesodermal progenitor cells generate notochord and floorplate (ventral spinal cord) (Row et al., 2016) and in both human and mouse embryonic stem cells and zebrafish tailbud stem cells, bipotential neuromesodermal progenitors generate paraxial mesoderm and posterior neural tube (Gouti et al., 2014; Martin and Kimelman, 2012). Even in the classical mesendoderm model, that is the C. elegans EMS cell, the MS (mesoderm) lineage also gives rise to some neurons (Sulston and Horvitz, 1977; Sulston et al., 1983). The lateral E cells of Ciona are also not yet fate-restricted to endoderm fate. At the 64-cell stage of development, the lateral E cell divides into one endoderm and one trunk lateral cell (mesenchyme) precursor, following induction of trunk lateral cell fate (Shi and Levine, 2008). Thus, as in other species, ascidian germ layer segregation is an progressive process (Tzouanacou et al., 2009) and NNE specification should thus be considered as its first step.

Regulatory architectures of mesendoderm

We have shown that, in ascidian embryos, individual mesendoderm lineage specifiers are required for the initiation of both mesoderm and endoderm GRNs (Figure 6). Furthermore, we have shown that the combinatorial activity of just three NNE factors is sufficient to reprogramme developing ectoderm cells to a mesendoderm state. The mesendoderm regulatory state in ascidian embryos is similar to the situation in the C. elegans EMS cell in which the MED1/2 GATA factors feed into both E (endoderm) and MS (mesoderm) lineage specification, such that MED1/2 directly activates both MS and E target genes (Broitman-Maduro et al., 2005; Maduro et al., 2001, 2015; McGhee, 2013). Similarly, Foxa.a and Foxd can bind to the upstream sequences of both Zic-r.b (NN lineage) and Lhx3/4 (E lineage), suggesting that this genetic interaction is direct (Kubo et al., 2010). While there is little doubt that mesendoderm transiently forms during embryogenesis of many animal models, and that both mesoderm and endoderm are induced by similar upstream regulators (β-catenin in invertebrates, β-catenin and Nodal in vertebrates), in most cases the transcriptional nature of the mesendoderm state does not appear to be similar to that of ascidians or nematodes. In particular, the existence of mesendoderm lineage specifiers (that is individual factors required for the initiation of both mesoderm and endoderm GRNs) have not been described in the majority of model organisms. For example, in sea urchins and anamniote vertebrates, mesendoderm has been described as a mixed regulatory state with simultaneous activation of mesoderm and endoderm GRNs, prior to the lineage segregation of these fates (Peter and Davidson, 2010; Rodaway and Patient, 2001). This type of ‘mixed-lineage’ regulatory architecture is also described in other systems and displays characteristics of multi-lineage priming, whereby the GRNs of two lineages are simultanously activated prior to lineage segregation (Figure 6c) (Graf and Enver, 2009; Nimmo et al., 2015). If this were the scenario for the ascidian mesendoderm regulatory state, one would expect individual NNE factors to be required for, and be able to induce, only one or other of the two subsequent lineages (NN or E), but not both. A ‘mixed-lineage’ regulatory architecture is therefore not consistent with our data describing the NNE mesendoderm regulatory state (Figure 6).

These two regulatory architectures are, however, unlikely to be mutually exclusive. In sea urchin and sea stars, for example, genes interacting with both mesoderm and endoderm GRNs have been identified (http://sugp.caltech.edu/endomes/) (Davidson et al., 2002; McCauley et al., 2015). It cannot be ruled out that mesendoderm lineage specifiers, acting upstream of both endoderm and mesoderm GRNs, are more broadly utilised, but are simply difficult to uncover due to the sheer complexity of early embryos and their GRNs (Ben-Tabou de-Leon and Davidson, 2009; Kiecker et al., 2016; Tremblay, 2010). It is also possible that the regulatory architecture of nematode and ascidian mesendoderm resulted from an adaption to a lineage-based mode of development with small numbers of cells, perhaps enabling these rapidly developing embryos to bypass the need for cross-repression and prolonged stabilisations of the endoderm and mesoderm GRNs. In summary, it is not yet clear whether an obligate mesendoderm state (that is a state with mesendoderm lineage specifiers) is present in the majority of bilaterian developmental programmes, although this seems to be the case in nematode and ascidian embryos.

Materials and methods

Overexpression and knockdown tools

Morpholinos (MOs) were purchased from GeneTools (Philomath, Oregon) and have been reported previously: β-catenin-MO (Hudson et al., 2013); Foxa.a-MO and Foxd-MO (Imai et al., 2006); Fgf9/16/20-MO and Fgf8/17/18-MO (Yasuo and Hudson, 2007), ETS1/2-MO (Bertrand et al., 2003). Foxa.a-MO was injected at 0.85 mM and ETS1/2-MO at 0.75 mM. All other morpholinos were injected at 0.5 mM. U0126 was used at 2 μM and BIO (GSK-3 inhibitor IX) at 2.5 μM (both were purchased from Calbiochem (Merck, Darmstadt, Germany)). Since a full-length cDNA clone for Ciona intestinalis Foxd is not available in gene collection plates (Gilchrist et al., 2015; Satou et al., 2002), we synthesised the Ciona savignyi Foxd mRNA from pRN3-Cs-Foxd (Imai et al., 2002b) using mMESSEGEmMACHINE kit (Thermo Fisher Scientific, Waltham, MA). The Ciona savigni Foxd used in this study corresponds to Genbank accession number AB057738.1. Foxd mRNA was injected at 75ng/μl. In order to generate pFT>Foxa.a, pFT>Foxd and pFT>Fgf9/16/20, we first constructed Gateway (Invitrogen, a brand of Thermo Fisher Scientific) pENTR clones containing ORFs of these genes. ORFs of Cs-Foxd, Foxa.a and Fgf9/16/20 were PCR-amplified using the following primer pairs and templates: Foxa.a-attB1 (aaaaagcaggctaccATGATGTTGTCGTCTCCACC) and Foxa.a-attB2 agaaagctgggtTTAGCTTGCTGGTACGCAC) on cicl044j20 template; FGF9-attB1 (aaaaagcaggctaccATGTCTATGTTAACCAACATGTTAGG) and FGF9-attB2 (agaaagctgggtTCAGTAGAGTCGGCCAGAGTAC) on citb007k01; CsFoxd-attB1 (aaaaagcaggctaccATGACTGTGGACTCTTGTACAG) and CsFoxd-attB2 (agaaagctgggtCTAAATAAGTTTATACGGGAATGG) on pRN3-Cs-Foxd. The Fucosyltransferase-like driver has been reported previously (Pasini et al., 2012). The promoter region was PCR-amplified using the following pair of primers to generate a destination vector pSP1.72BSSPE-pFT::RfA-venus (Roure et al., 2007): pFT-attB3 (ggggacaagtttgtataataaagtaggctGGCATCATAACGTACAACCTG) and pFT-attB5 (ggggaccactttgtatacaaaagttgggtTGCAGCGGTAGAGTTTACTATTATC). pFT>Foxa.a, pFT>Foxd and pFT>Fgf9/16/20 were then generated by LR reaction between corresponding pENTR clones and pSP1.72BSSPE-pFT::RfA-venus.

Embryological experiments

Adult Ciona intestinalis were purchased from the Station Biologique de Roscoff (France). Blastomere names, lineage and the fate maps are previously described (Conklin, 1905; Nishida, 1987). Ascidian embryo culture and microinjection have been described (Sardet et al., 2011). All microinjections were carried out in unfertilised eggs. The electroporation protocol was based on Christiaen et al., 2009. Up to 60 μg of circular plasmid DNA was made up to 250 μl at 0.6 M mannitol. DNA/mannitol solution was mixed with 100 μl of eggs in artificial sea water supplemented with 0.5% BSA (to help prevent sticking). Electroporation was carried out at 50V for 16 ms using a BTX (Harvard Apparatus, Holliston, Massachusetts) ECM 830 and electroporated embryos transferred to agarose-coated dishes. For data shown in Figure 5—figure supplement 1a, 50 μg of pFT>Foxd was used. Otherwise, each FT construct was used at 20 μg to give a maximum total of 60 μg. pFT>tdTomato was used as a control electroporation at 60 μg. In all experiments, embryos that failed to develop were discarded and all other embryos scored. All data were pooled from at least two independent experiments (i.e., on different batches of embryos).

The experimental design of the BIO treatment of electroporated ectodermal explants, shown in Figure 5c, is as follows. Embryos were electroporated with three plasmids, pFT>Foxa.a + pFT>Foxd + pFT>Fgf9/16/20. Ectoderm explants were isolated at the eight-cell stage from control (unelectroporated) and electroporated embryos. Each sample of explants was split into two groups and one group of each treated with BIO from the 76-cell stage. BIO treatment was continued until fixation, when sibling embryos reached the mid-gastrula stage. Explants remained in BIO for approximately 1 hr at 20°C.

In situ hybridisation, gene naming, alkaline phosphatase staining and dpERK and β-catenin immunofluorescence

All gene markers used for in situ hybridisation have previously been described (Hudson et al., 2013; Imai et al., 2004) (http://ghost.zool.kyoto-u.ac.jp). According to recent nomenclature guidelines, we used Zic-r.b (previously called ZicL) to describe the five copies of ZicL gene named Zic-r.b – Zic-r.f, Lhx3/4 (previously Lhx3) and Efna.d (previously ephrin-Ad) (Stolfi et al., 2015). The in situ hybridisation and alkaline phosphatase staining protocols are previously described (Hudson et al., 2013). All single embryo panels, except those in Figure 3c, were mounted in 50–80% glycerol and photographed on an Olympus (Tokyo, Japan) BX51 using a Leica DFC310FX camera. All multi-embryo panels as well as single-embryo panels in Figure 3c were taken of embryos in PBT on a Leica (Leica microsystems, Vienna, Austria) Macroscope Z16 APO with a Canon (Tokyo, Japan) EOS 60D camera. The dpERK and β-catenin immunofluorescence protocols are described previously (Haupaix et al., 2014; Hudson et al., 2013). Immunostained embryos were mounted in Vectashield-DAPI (Vector laboratories, Burlingame, CA), analysed on a Leica SP5 confocal microscope and processed with Image J.

Acknowledgements

We thank U Röthbacher and P Lemaire (FT promoter), H Nishida (β-catenin antibody), Y Satou (pRN3-Cs-Foxd and Foxa.a-MO) and N Satoh (Gene Collection Plates) for tools, Evelyn Houliston and David McClay for critical reading of the manuscript and helpful discussions and Jenifer Croce and David McClay for interesting discussions on sea urchin mesendoderm GRNs.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

Centre National de la Recherche Scientifique to Hitoyoshi Yasuo.
Université Pierre et Marie Curie to Hitoyoshi Yasuo.
Fondation ARC pour la Recherche sur le Cancer #1144 to Hitoyoshi Yasuo.
Agence Nationale de la Recherche ANR-09-BLAN-0013-01 to Hitoyoshi Yasuo.
Fondation ARC pour la Recherche sur le Cancer PJA 20131200223 to Hitoyoshi Yasuo.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

CH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents.

CS, Acquisition of data, Analysis and interpretation of data.

HY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents.

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eLife. 2016 Jun 28;5:e14692. doi: 10.7554/eLife.14692.015

Decision letter

Editor: Alejandro Sánchez Alvarado¹

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "An essential transient mesendoderm state during germ layer segregation in ascidian embryos" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Janet Rossant as the Senior Editor. One of the two reviewers has agreed to reveal his identity: Brad Davidson.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Specifically, while the study is carefully carried out with nice experimental findings here, the reviewers felt that the overall hypothetical framework of a specific mesendodermal state was not well supported. Given this, the paper does not fundamentally change the way we think about the evolution of developmental regulatory networks and is perhaps more suited to a specialized developmental journal.

Reviewer #1:

Hudson et al. present a re-evaluation of established regulatory relationships between early regulators of endomesodermal fates in early ascidian embryo, using Ciona as a model and focusing on 32- and 64-cell stages.

The main point of the paper is to argue that FoxD, FoxAa and Fgf9/16/20 together form a mesendoderm-specific regulatory state activated downstream of β-catenin. One of the key novel experimental merits of the paper is to show that this cocktail of regulators is sufficient to reprogram ectoderm precursors into mesendodermal tissues, whether the mesodermal notochord or the endoderm, providing prolonged β-catenin activity for the latter.

Even though the experiments are well executed and reasonably interpreted, I have concerns about the whole premise of the paper:

It is difficult to call NNE precursors strictly mesendoderm, since the NN precursor will also produce posterior nerve chord. Rather it seems likely that these early progenitors are quite ascidian-specific even though they use conserved pan-bilaterian regulatory inputs such as the β-catenin/TCF regulatory axis.

For this cocktail of regulators to be specific to a mesendoderm state, one would expect them to be restricted to the pluripotent stage of the NNE progenitor. This seems to be the case for FoxD, however Fgf9/16/20 is later maintained in the NN progenitors and notochord, whereas FoxAa is later maintained in the endoderm. This suggests that – as shown to be the case for NN by Kobayashi et al., Genesis, 2013 – the NNE progenitors are primed by expressing early regulators of both the NN and the E fate. Thus, I am not convinced that the NNE "mesendoectoderm" progenitors depart significantly from a more classical model of multilineage priming of progenitors, as stated by the authors.

Finally, by the authors' admission, several of these regulatory linkages have been documented before, so it is not always clear what the novel insights really are.

Reviewer #2:

In this manuscript, the authors make substantial progress in delineating the transcriptional circuitry underlying mesendoderm lineage specification. Through an extensive and well-documented series of manipulations the authors demonstrate that three transcription factors are both necessary and sufficient for establishing the tunicate mesendodermal lineage. The authors then attempt to employ their results to help resolve questions about the nature and evolution of mesendodermal regulatory states in various clades. However, some of these interpretations are unclear and potentially inaccurate as discussed below.

My primary concern regards the overall framework the authors used to interpret their results. In the Abstract, the authors propose the mesendoderm may represent an "obligatory" or "essential" developmental state and indicate that their data supports this assertion. However, it is not clear that this is an accurate assessment.

According to this premise, the loss of the mesendoderm regulatory state should preclude the subsequent establishment of endoderm and mesodermal lineages. Yet, the authors’ data contradict this. Instead they found that loss of one or more key mesendodermal regulatory factors did not preclude the eventual establishment of endoderm or mesoderm. In particular, they showed that:

a) ZicL (a mesoderm lineage marker) expression was eventually restored after inhibition of FGF signaling (Figure 2);b) Endoderm differentiation (assayed by alkaline phosphatase activity) occurred despite inhibition of FGF or FoxD (Figure 2).

They also found that single "mesendoderm" factors were able to restore either mesoderm or endoderm even though were not able to restore the mesendoderm state, effectively bypassing this state and demonstrating that it is not essential or obligatory. In particular they showed that:

FoxD alone was able to restore expression of Mesoderm lineage genes after loss of the mesendoderm regulatory state through disruption of B-catenin (Figure 4).

There are at least three ways that the authors could address these concerns.

1) Clarify what they mean by an "essential" role for the mesendoderm and how their experimental results support this hypothesis.

2) Modify their hypothesis, possibly by suggesting that the three mesendodermal factors are locked into a recursive regulatory relationship. This caveat might explain why disruption of one factor only temporarily blocks the establishment of this regulatory state (Figure 2). It may also serve to explain why expression of a single factor is sufficient to re-establish the mesendodermal state/subsequent germ layer sub-specification (Figure 3). If they do propose this explanation, they should also evaluate whether it is supported by any data.

3) Discard their current hypotheses and focus on an alternative framework. For example, they could choose to focus their interpretation on different regulatory architectures that might underlie mesendoderm regulatory states in different embryos and some potential functional implications of these different architectures. This framework may allow a more productive comparison to other characterized endomesodermal circuits (nematode/sea urchins) as further discussed in the next point.

A secondary concern focuses on the authors' statement that a "distinct mesendodermal state" is not present in sea urchins (Introduction, first paragraph and Discussion, second paragraph). The sea urchin and sea star GRNs both include a conserved, transient mesendodermal state based on B-catenin dependent expression of wnt8 and Otx (Hinman, Yankura and McCauley 2009). The cited paper (Peter and Davidson, 2010) does state that:

"In a strict sense, early endomesoderm specification therefore involves the concomitant activation of two separate GRNs, rather than the activation of a common endomesoderm GRN which diverges into two daughter programs. The co-expression of regulatory genes to which we refer to as endomesoderm regulatory state is the result of these two GRNs."

However, this interpretation is not definitive and a good argument could be made that the endomesodermal state is established prior to the simultaneous activation of the two germ-layer specific programs, as laid out in the cited Hinman paper. The authors should clarify exactly what they think defines a distinct endodermal regulatory state and whether or not their data suggests a tunicate regulatory circuitry that is fundamentally distinct from that observed in sea urchins/sea stars.

eLife. 2016 Jun 28;5:e14692. doi: 10.7554/eLife.14692.016

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

[Please also refer to: “Finally, by the authors' admission, several of these regulatory linkages have been documented before, so it is not always clear what the novel insights really are.”]

This comment is in striking contrast to reviewer 2 who describes our work as ‘substantial progress’. Importantly, the point of our manuscript was to characterise the mesendoderm lineage specifiers and reveal exactly how these factors provide the ground state for the second round of β-catenin driven binary fate decisions that segregates the NN and E lineages and it was not possible to ascertain this from the data already published. A summary of the published data is summarised below and is described more carefully in the revised version of our manuscript.

In the literature at the time of our first submission it was reported that Foxa.a, Foxd and Fgf9/16/20 are downstream of β-catenin and that Foxd is likely to be direct. Data showing that Foxa.a is a transcriptional target of β-catenin had never actually been documented, though it was previously mentioned in Imai et al., 2002b. The relationship between β-catenin and any of the three factors was also not reported at the 16-cell stage in relation to either loss or activation (BIO-treatment) of β-catenin. It was important to perform this analysis at the 16-cell stage, prior to fate segregation of the mesendoderm lineage into mesoderm and endoderm lineages at the 32-cell stage. While this data at the time was therefore novel (albeit expected), it has since shown by the group of Yutaka Satou that all three of these factors are downstream targets of β-catenin with at least Fgf9/16/20 and Foxd being direct targets (Oda-Ishii et al., PLoS Genetics, 2016). This highly relevant manuscript is duly discussed in the revised version of our manuscript. We preferred to retain the, now mostly confirmatory, data in Figure 1 of our revised manuscript as it forms an important basis for our study.

To summarise the published data on the role of Foxa.a, Foxd and Fgf9/16/20 in Ciona: Foxa.a was reported to be required for notochord and endoderm formation; Foxd for notochord but not endoderm and Fgf9/16/20 for notochord (partially) but not endoderm (Imai et al., 2002a, 2002b, Imai et al., 2006; Yasuo and Hudson, 2007). Similarly, in Halocynthia embryos Foxa.a, Foxd and FGF-signals were reported to be required for notochord formation (Kumano et al., 2006).

Important for our study was whether the expression of the NN and E lineage specifiers of the 32-cell stage embryo (Zic-r.b and Lhx3/4 respectively) was affected by loss of any of the NNE factors in Ciona. However, only an examination of Zic-r.b expression at the 32-cell stage following Foxd-MO injection has been reported previously (Imai et al., 2002b). Thus, it was not known prior to our study whether Foxa.a, Foxd or FGF9/16/20 were involved in the correct initiation of NN and E cell genetic programmes. Thus, our data showing that all three factors are required individually for the initiation of gene expression in both NN and E cells is novel and also quite unexpected. Much of the supporting data, showing that endoderm gene expression is repressed at least until the early gastrula stage following Foxd or FGF-signal inhibition is also novel. The demonstration that Foxd and FGF-signals act in a partially redundant way during late endoderm specification is also novel, these factors were previously deemed dispensable for endoderm formation. Finally, the conversion of animal cells to an NNE-like state by co-expression of the three NNE factors, together with the demonstration that developing ectoderm cells can be reprogrammed to mesendoderm fate by co-expression of these three factors are novel.

In summary, while we agree that the regulatory relationships of these factors were partially known, the available data was not sufficient to answer our particular question and required further investigation. This should not be interpreted in any way as a criticism of the published manuscripts, which were addressing distinct questions to ours.

Even though the experiments are well executed and reasonably interpreted, I have concerns about the whole premise of the paper:

We agree with the reviewer that the NNE precursors are not strictly mesendodermal. However, we do not agree that the ascidian ‘neuro-mesendodermal’ cells are likely to be ascidian specific. Rather, a study of the literature reveals common origins for neural and mesoderm in many species and as in many other species, ascidian mesendoderm specification begins with β-catenin signalling. The ascidian NNE (Neural, Notochord, Endoderm) cell, specified by β-catenin signals, divides into a neuro-mesoderm (NN) and (mostly) endoderm (E) progenitor. The NN cell generates notochord (mesoderm) and the posterior part of the CNS, including the equivalent of the ‘spinal cord’ with its ventral cells that are potentially equivalent to the vertebrate floor plate. Formation of these tissues follows a binary cell fate choice between neural and notochord: not neural and epidermis. Similarly, in the zebrafish tailbud, bipotential progenitor cells generate notochord and floorplate (Row et al., 2016, Development); in both human and mouse embryonic stem cells and zebrafish tailbud stem cells, bipotential neuromesodermal progenitors generate paraxial mesoderm and posterior neural tube (Gouti et al., PLOS Biology, 2014; Martin and Kimelman, 2012; Dev. Cell.). Even in the classical mesendodermal model, that is the C. elegans EMS cell, the MS lineage also gives rise to neurons (Sulston and Horvitz, 1977, Developmental Biology; Sulston et al., 1983 Developmental Biology). Further, several mutations in mice result in fate changes between neural and mesoderm- Fgfr1 mutant cells fail to pass through the primitive streak and form secondary neural tubes instead of mesoderm (Ciruna et al., 1997, Development); in Wnt-3a mutants, cells ingress through the primitive streak, but fail to form somites, instead forming secondary neural tubes (Yoshikawa et al., 1997, Developmental Biology); in Tbx6 mutants posterior somites are converted to extra neural tubes (Chapman and Papaioannou, 1998, Nature). While classically the mesoderm germ layer is called as such rather than neuromesoderm, we feel that it is highly likely, due to the widespread nature of the neuro-mesoderm bipotential choice, that some neural fates originates from the so-called the ‘mesoderm’ germ layer.

In response to this concern raised by reviewer 1, we have now discussed in more detail the process of germ layer segregation in ascidian embryos to make it clear that 1) it is a progressive process, 2) part of the neural tissue shares a common origin with mesoderm, and 3) these two points are similar to what is observed in other chordate embryos.

We disagree. Firstly, we see absolutely no reason that a regulator can only be used once rather than for multiple rounds of cell lineage decisions (though this would be more convenient for developmental biologists). Importantly for our study it is the combinatorial activity of these three factors that specifies the NNE state and co-expression of all three factors takes place only at the 16-to-32-cell stages of development in mesendoderm lineages. The on-going expression of these factors in different lineages does not preclude their role in NNE cell specification, but rather suggests pleiotropic requirements. Indeed, in order to unravel the role of NNE factors at the 16-cell stage from their later roles, we focused on the effect of their loss at the 32-cell stage, that is one cell cycle after the onset of NNE factor expression. The 32-cell stage corresponds to the stage when the lineage segregation of notochord-neural (NN) and endoderm (E) takes place. Later stages were also addressed, in part to tie in our data with the published literature. What our data showed was that all three factors were required individually for the correct initiation of both NN and E cell genetic programmes. This concern prompted us to highlight that these factors likely play on-going roles during NN and E lineage specification.

We also disagree that the NNE cells are an example of multi-lineage priming. We found no evidence for this. If NNE cell fate specification were a case of multi-lineage priming, one would predict that loss of individual NNE factors (Foxa.a, Foxd or Fgf9/16/20) would result in loss of either NN or E gene expression. However, (initially to our surprise) both NN and E gene expression was reduced if any one of these factors was inhibited. We concluded from this that NNE factors are not indicative of a primed state. In support of this proposition, overexpression of any one factor in developing ectoderm lineages was unable to promote ectopic mesoderm fate, whereas overexpression of all three factors could. This is again not what would be predicted if NNE cells were primed with a mixture of NN and E specifiers: in this case overexpression of individual NNE factors would promote either NN fates (for the NN factors) or E fates (for the E factors). In our system, it is clearly β-catenin and not cross-antagonism of primed NN and E factors, which generates the differential fate decision between NN and E cells.

Issues raised by both reviewers made it clear to us that we had not been sufficiently clear with what we meant by a ‘distinct’ regulatory state and how this differed from a ‘mixed' or 'mixed-lineage’ regulatory state. A ‘mixed-lineage’ regulatory state shares characteristics to a primed regulatory state whereby a mixture of cell-type specific genes are transcriptionally activated prior to their restricted expression in a cell-type specific fashion following lineage segregation (e.g. Graf and Enver, 2009, Nature). To put it more simply, a cell ‘AB’ expresses factors that are, individually, required for either of the subsequent fates ‘A’ or ‘B’. We preferred, however, not to call this a ‘primed’ regulatory state, since in many cases priming involves lows levels of gene activation followed by reinforcement in a lineage specific manner. Instead, we used the term ‘mixed-lineage regulatory state’ a term previously employed (Guo et al., Dev. Cell, 2010). A ‘distinct’ regulatory state would be, in contrast, a situation wherein the cell ‘AB’ expresses factors (AB lineage specification factors) and each of them is required for both subsequent states A and B. We realised that ‘distinct’ was perhaps not the best word, since a ‘mixed-lineage’ regulatory state is also distinct from either of the subsequent lineage-specific states. In the revised manuscript, we decided to avoid the use of the term ‘distinct regulatory state' and instead compare and contrast the regulatory situation of ascidians to that described in other systems. Our manuscript now includes a clear definition of a ‘mixed-lineage regulatory state’ with supporting schematics (Figure 6). We hope that the schematics more clearly highlight the difference between regulatory architectures of ascidian mesendoderm regulatory state and a mixed-lineage regulatory state. We also decided to no longer bring this point up in the Introduction, but rather to focus this, now more in depth comparison, to the Discussion section.

To summarise our findings, we identified in Ciona the mesendoderm lineage specifiers, whose combinatorial activity is sufficient to reprogramme developing ectoderm cells to a mesendoderm state. This ectopic mesendoderm state segregates further into either mesoderm or endoderm lineages depending on β-catenin activity, in a similar way to the endogenous mesendoderm lineage.

Finally, by the authors' admission, several of these regulatory linkages have been documented before, so it is not always clear what the novel insights really are.

See response to the first comment.

Reviewer #2:

a) ZicL (a mesoderm lineage marker) expression was eventually restored after inhibition of FGF signaling (Figure 2);

b) Endoderm differentiation (assayed by alkaline phosphatase activity) occurred despite inhibition of FGF or FoxD (Figure 2).

We understand the reviewer’s concern. What we meant by ‘essential’ was ‘essential for normal specification’. Indeed, NN and E cells fail to correctly initiate their genetic programmes when any one of the three NNE factors is inhibited. We accept that perhaps our use of the word 'essential' was too enthusiastic since there is likely to be some regulative processes that lead to the recovery of tissues under certain conditions. Below, we respond to the particular points raised in the first concern and explain why we maintain that the NNE factors are required for correct mesendoderm formation.

Following Fgf9/16/20 inhibition, Zic-r.b expression is restored at the 64-cell stage in both notochord and neural precursors. However, this is not that surprising considering that while FGF-signalling is required for notochord fate, it is not compatible with neural fate (differential ERK activation between notochord and neural fates drives their binary fate choice). Thus, one would expect an FGF-independent expression of Zic-r.b at the 64-cell stage, at least in neural fated cells. Consistent with potentially independent gene regulation between the 32- and 64-cell stages, it has been shown that Zic-r.b expression at these stages can be mediated by separate enhancer elements (Anno et al., 2006, Genes Dev. Evol.). In the revised version of our manuscript, we described in more detail the regulation of Zic-r.b and the on-going role of FGF signals during the correct specification of the notochord lineage. While, it is not possible to ascertain whether FGF-signalling is absolutely required at each successive time point, we would nonetheless argue that FGF-signalling via ERK is involved during the step-by-step (Zic-r.b at 32-cell, Bra at 64-cell) specification of notochord.

In Foxd-MO or Fgf9/16/20-MO injected embryos, while endoderm gene expression is reduced, at least until the early gastrula stage, endoderm fate eventually recovers by larval stages. Thus, while we are confident that Foxd and Fgf9/16/20 are required for the correct specification of endoderm, we also agree that endoderm fate can recover. This kind of recovery is not unusual. In sea urchin for example, in the absence of pmar1, skeletogenic mesenchyme fate recovers as a result of the action of blimp1, a process coined ‘regulative recovery’ (Smith and Davidson, 2009; PNAS). To use an ascidian example, the loss of Fgf9/16/20 results in an early loss of the notochord genetic programme. However, notochord cells later recover to some extent via the action of Fgf8/17/18 (Yasuo and Hudson, 2007). To turn the argument around, we would not argue from this that Fgf9/16/20 is not required for notochord formation, it clearly is required for notochord cells to follow their correct developmental programme. In the current study, the data showing that loss of Foxd and Fgf9/16/20 in combination leads to a strong loss of endoderm supports a level of 'redundancy' between these two factors, but also a requirement. While we do not find the recovery of endoderm particularly worrying for our model, we do understand the reviewer’s concern. We have now been more careful to highlight our observation that the NNE factors are required for the correct initiation of NN and E lineages, with some recovery during later development. We also no longer refer to an 'essential' regulatory state.

FoxD alone was able to restore expression of Mesoderm lineage genes after loss of the mesendoderm regulatory state through disruption of B-catenin (Figure 4).

No, this is not correct. Foxd is not alone in this experiment. As shown in Figure 1B, loss of β-catenin results in quite broad expression of Foxa.a in both a-line cells as well as A-line cells (which are now most likely converted to an a-line fate based on co-expression of Foxa.a and Efna.d(Figure 2)). On the other hand, Foxd injection results in loss of Efna.d (a potent inhibitor of FGF-MEK-ERK-signals) and weak activation of Fgf9/16/20 (Figure 3—figure supplement 1). Thus, in β-catenin-MO injected embryos, injection of Foxd into the egg restores the co-expression of all three factors by the 16-cell stage. In support of this hypothesis, treatment of β-catenin-MO+Foxd mRNA embryos with UO126 to inhibit FGF-signalling results in a similar reduction of Zic-r.b and Bra as is seen in control embryos. We agree that we did not explain this experiment very well in our previous manuscript and in response to this concern of Reviewer 2, we now explained this experiment in detail.

Finally, in support of our hypothesis that all three factors are required to induce an NNE-like state, our overexpression data clearly show that all three factors are required to reprogramme ectoderm into mesendoderm (Figure 5).

There are at least three ways that the authors could address these concerns.

1) Clarify what they mean by an "essential" role for the mesendoderm and how their experimental results support this hypothesis.

We agree with Reviewer 2 that this was not sufficiently clear in our submitted manuscript. In response to both reviewers, we now include an explicit description of different regulatory architectures.

We think that the reviewer is suggesting that the three NNE factors may be locked into a kind of regulatory ‘kernel’ whereby each factor induces the expression of the others? This is a nice idea, but it is not supported by our data. Inhibition of any one factor does not affect expression of any other (Figure 2—figure supplement 1), although injection of Foxd does induce low levels of Fgf9/16/20 expression. Most importantly for our conclusions, expression of a single factor is not sufficient to re-establish NNE fate in developing ectoderm cells, as described above.

Although we did not frame the question specifically in these terms, we were indeed trying to compare the architecture of the initial mesendoderm GRN of ascidians to that of different embryos. The specific aim of our study was to identify the mesendoderm lineage specifiers of the ascidian embryo and reveal how they influence the outcome of the second β-catenin driven binary fate decision that segregates the NN and E lineages. We concluded that ascidian mesendoderm bears most similarity to that in the C.elegans model and is not based on a mixed-lineage regulatory architecture. To date, to our knowledge, no mesendoderm lineage specifiers (that is individual factors at the top of the genetic hierarchy that are required for initiation of both mesoderm and endoderm GRNs) have been identified in other systems, other than the inducers (β-catenin and Nodal) themselves. However, the reviewer is correct to say that the published literature does not exclude the existence such mesendoderm specifiers upstream of both mesoderm and endoderm GRNs in sea urchins and vertebrates. It may simply be that regulative recovery/redundancy, together with extremely complicated early development and GRNs make the discovery of these kinds of factors very difficult. Or it may be that ascidians (and nematodes) do indeed use a fundamentally distinct mode of mesendoderm specification due to the simplification and rapidity of their early development. In the revised version of our manuscript, we attempt to address all of these issues in more detail.

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PERMALINK

Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos

Clare Hudson

Cathy Sirour

Hitoyoshi Yasuo

Roles

Abstract

Introduction

Figure 1. Foxa.a, Foxd and Fgf9/16/20 are candidate NNE lineage specification factors.

Results

Foxa.a, Foxd and Fgf9/16/20 are nβ-catenin transcriptional targets in NNE cells

Foxa.a, Foxd and Fgf9/16/20-signals are required for the correct initiation of both NN and E gene expression

Figure 2. Foxa.a, Foxd and Fgf9/16/20 are required for initiation of NN and E gene expression.

Figure 2—figure supplement 1. Foxa.a, Foxd and Fgf9/16/20 are required for initiation of NN and E gene expression.

Figure 2—figure supplement 2. Endoderm formation under various conditions.

Table 1.

Combinatorial activity of Foxa.a, Foxd and Fgf9/16/20 induces a mesendoderm state

Figure 3. Creation of an ectopic Foxa.a, Foxd, FGF-signal overlap leads to ectopic mesendoderm formation.

Figure 3—figure supplement 1. Foxd mRNA injection leads to repression of Efna.d and upregulation of Fgf9/16/20 in ectodermal cells at the 16-cell stage.

Figure 4. Foxd mRNA injection rescues mesoderm in β-catenin-MO injected embryos.

Foxa.a, Foxd and Fgf9/16/20 act synergistically to reprogramme developing ectoderm cells to a mesendoderm state

Figure 5. Reprogramming the ectoderm lineage to mesendoderm.

Figure 5—figure supplement 1. Reprogramming of ectoderm cells to mesendoderm fates.

Figure 5—figure supplement 2. Confirmation that BIO-treatment of ectoderm explants at the 76-cell stage results in nuclear localisation of β-catenin.

Discussion

Figure 6. Gene regulatory model for segregation of NNE into NN and E lineages.

A model for ascidian germ layer segregation

Regulatory architectures of mesendoderm

Materials and methods

Overexpression and knockdown tools

Embryological experiments

In situ hybridisation, gene naming, alkaline phosphatase staining and dpERK and β-catenin immunofluorescence

Acknowledgements

Funding Statement

Funding Information

Additional information

Competing interests

Author contributions

References

Decision letter

Roles

Author response

ACTIONS

PERMALINK

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